Single-particle fluorescence microscopy of biological assembly reactions is emerging in its potential to obtain information that is lost during the ensemble averaging of conventional biochemical analysis. However, efforts to realize this potential have stalled at the level of the specimens used. The Pi's laboratory has been developing the needed specimen preparation and accompanying procedures. For continuous real time single-particle visualization of assembly, the Pi's laboratory has found specimen preparation procedures for concentrating/restricting comparatively large (-30 nm in radius) particles in a thin zone of solution next to a cover glass. However, similar conditions for smaller particles (single protein molecules) are not yet developed. The specific aims are the following: (1) For single-particle fluorescence microscopy, specimen preparation procedures will be developed that achieve (a) concentration of comparatively small particles, including monomeric proteins, in a thin zone of solution next to a cover glass, and (b) retention by these particles of thermal motion. (2) Procedures of single-particle fluorescence microscopy will be developed for observing and analyzing both dimerization and more complex events of the assembly of macromolecules. In so doing, determination will be made of whether our single-particle procedure yields dissociation constants that agree with the dissociation constants measured by use of conventional, ensemble averaging procedures. Analysis will be performed of complex viral assembly. Achievement of these aims will make the assembly of multimolecular complexes routinely accessible to analysis by single-particle fluorescence microscopy. Applications are anticipated in the analysis of the assembly of disease-causing viruses and determining pathways of oncogenesis.